Methods and Compositions for Weed Control

ABSTRACT

The present invention provides novel compositions for use to enhance weed control. Specifically, the present invention provides for methods and compositions that modulate glutamine synthetase in weed species. The present invention also provides for combinations of compositions and methods that enhance weed control.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 13/612,948, filed on Sep. 13, 2012, which claimsthe benefit of priority of U.S. Provisional Application No. 61/534,076,filed on Sep. 13, 2011, both of which are incorporated in their entiretyherein by reference.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of a sequence listing is filed with thisapplication by electronic submission and is incorporated into thisapplication by reference in its entirety. The sequence listing iscontained in the file named P34113US02_SEQ.txt, which is 849,269 bytesin size (measured in operating system MS windows) and was created onJul. 28, 2016.

FIELD

The methods and compositions generally relate to the field of weedmanagement. More specifically, related to glutamine synthetase (GS)genes in plants and compositions containing polynucleotide molecules formodulating their expression. Further provided are methods andcompositions useful for weed control.

BACKGROUND

Weeds are plants that compete with cultivated plants in an agronomicenvironment and cost farmers billions of dollars annually in crop lossesand the expense of efforts to keep weeds under control. Weeds also serveas hosts for crop diseases and insect pests. The losses caused by weedsin agricultural production environments include decreases in crop yield,reduced crop quality, increased irrigation costs, increased harvestingcosts, reduced land value, injury to livestock, and crop damage frominsects and diseases harbored by the weeds. The principal means by whichweeds cause these effects are: 1) competing with crop plants for water,nutrients, sunlight and other essentials for growth and development, 2)production of toxic or irritant chemicals that cause human or animalhealth problem, 3) production of immense quantities of seed orvegetative reproductive parts or both that contaminate agriculturalproducts and perpetuate the species in agricultural lands, and 4)production on agricultural and nonagricultural lands of vast amounts ofvegetation that must be disposed of Herbicide tolerant weeds are aproblem with nearly all herbicides in use, there is a need toeffectively manage these weeds. There are over 365 weed biotypescurrently identified as being herbicide resistant to one or moreherbicides by the Herbicide Resistance Action Committee (HRAC), theNorth American Herbicide Resistance Action Committee (NAHRAC), and theWeed Science Society of America (WSSA).

The glutamine synthetase (GS) enzyme is an essential enzyme in themetabolism of nitrogen by catalyzing the condensation of glutamate andammonia to form glutamine. This enzyme is the target of phosphinic acidsherbicides that include glufosinate-ammonium and bialaphos.

SUMMARY

In one aspect, the invention provides a method of plant controlcomprising an external application to a plant of a compositioncomprising a polynucleotide and a transfer agent, wherein thepolynucleotide is essentially identical or essentially complementary toa glutamine synthetase (GS) gene sequence or fragment thereof, or to theRNA transcript of said GS gene sequence or fragment thereof, whereinsaid GS gene sequence is selected from the group consisting of SEQ IDNOs: 1-59 or a polynucleotide fragment thereof, whereby the weedy plantgrowth or development or reproductive ability is reduced or the weedyplant is made more sensitive to a GS inhibitor herbicide relative to aweedy plant not treated with said composition. In this manner, plantsthat have become resistant to the application of GS inhibitor containingherbicides may be made more susceptible to the herbicidal effects of aGS inhibitor containing herbicide, thus potentiating the effect of theherbicide. The polynucleotide fragment is at least 18 contiguousnucleotides, at least 19 contiguous nucleotides, at least 20 contiguousnucleotides or at least 21 contiguous nucleotides in length and at least85 percent identical to a GS gene sequence selected from the groupconsisting of SEQ ID NOs: 1-59 and the transfer agent is anorganosilicone composition or compound. The polynucleotide fragment canalso be sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA, ordsDNA/RNA hybrids. The composition can include more than onepolynucleotide fragments, and the composition can include a GS inhibitorherbicide and/or other herbicides (co-herbicides) that enhance the weedcontrol activity of the composition.

In another aspect, polynucleotide molecules and methods for modulatingGS gene expression in plant species are provided. The method reduces,represses or otherwise delays expression of a GS gene in a plantcomprising an external application to a plant of a compositioncomprising a polynucleotide and a transfer agent, wherein thepolynucleotide is essentially identical or essentially complementary toa GS gene sequence or fragment thereof, or to the RNA transcript of theGS gene sequence or fragment thereof, wherein the GS gene sequence isselected from the group consisting of SEQ ID NOs: 1-59 or apolynucleotide fragment thereof. The polynucleotide fragment is at least18 contiguous nucleotides, at least 19 contiguous nucleotides, at least20 contiguous nucleotides at least 21 contiguous nucleotides in lengthand at least 85 percent identical to a GS gene sequence selected fromthe group consisting of SEQ ID NOs:1-59 and the transfer agent is anorganosilicone compound. The polynucleotide fragment can also be senseor anti-sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids.Polynucleotide molecules comprising SEQ ID NOs: 37-1056 are fragments ofthe GS gene.

In a further aspect, the polynucleotide molecule containing compositionmay be combined with other herbicidal (co-herbicides) compounds toprovide additional control of unwanted plants in a field of cultivatedplants.

In a further aspect, the polynucleotide molecule composition may becombined with any one or more additional agricultural chemicals, suchas, insecticides, fungicides, nematicides, bactericides, acaricides,growth regulators, chemosterilants, semiochemicals, repellents,attractants, pheromones, feeding stimulants, biopesticides, microbialpesticides or other biologically active compounds to form amulti-component pesticide giving an even broader spectrum ofagricultural protection.

DETAILED DESCRIPTION

Provided are methods and compositions containing a polynucleotide thatprovide for regulation, repression or delay of GS (glutamine synthetase)gene expression and enhanced control of weedy plant species andimportantly GS inhibitor resistant weed biotypes. Aspects of the methodcan be applied to manage various weedy plants in agronomic and othercultivated environments.

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Where a term is provided in the singular, theinventors also contemplate aspects of the invention described by theplural of that term.

By “non-transcribable” polynucleotides is meant that the polynucleotidesdo not comprise a complete polymerase II transcription unit. As usedherein “solution” refers to homogeneous mixtures and non-homogeneousmixtures such as suspensions, colloids, micelles, and emulsions.

Weedy plants are plants that compete with cultivated plants, those ofparticular importance include, but are not limited to important invasiveand noxious weeds and herbicide resistant biotypes in crop production,such as, Amaranthus species A. albus, A. blitoides, A. hybridus, A.palmeri, A. powellii, A. retroflexus, A. spinosus, A. tuberculatus, andA. viridis; Ambrosia—A. trifida, A. artemisifolia; Lolium—L.multiflorum, L. rigidum, L perenne; Digitaria—D. insularis; Euphorbia—E.heterophylla; Kochia—K. scoparia; Sorghum—S. halepense; Conyza—C.bonariensis, C. canadensis, C. sumatrensis; Chloris—C. truncate;Echinochola—E. colona, E. crus-galli; Eleusine—E. indica; Poa—P. annua;Plantago—P. lanceolate, Avena—A. fatua; Chenopodium—C. album; Setariaspecies—S. viridis, Abutilon theophrasti, Ipomoea species, Sesbaniaspecies, Cassia species, Sida species, Brachiaria, species and Solanumspecies.

Additional weedy plant species found in cultivated areas includeAlopecurus myosuroides, Avena sterilis, Avena sterilis ludoviciana,Brachiaria plantaginea, Bromus diandrus, Bromus rigidus, Cynosurusechinatus, Digitaria ciliaris, Digitaria ischaemum, Digitariasanguinalis, Echinochloa oryzicola, Echinochloa phyllopogon, Eriochloapunctata, Hordeum glaucum, Hordeum leporinum, Ischaemum rugosum,Leptochloa chinensis, Lolium persicum, Phalaris minor, Phalarisparadoxa, Rottboellia exalta, Setaria faberi, Setaria viridis var,robusta-alba schreiber, Setaria viridis var, robusta-purpurea, Snowdeniapolystachea, Sorghum sudanese, Alisma plantago-aquatica, Amaranthuslividus, Amaranthus quitensis, Ammania auriculata, Ammania coccinea,Anthemis cotula, Apera spica-venti, Bacopa rotundifolia, Bidens pilosa,Bidens subalternans, Brassica tournefortii, Bromus tectorum, Camelinamicrocarpa, Chrysanthemum coronarium, Cuscuta campestris, Cyperusdifformis, Damasonium minus, Descurainia sophia, Diplotaxis tenuifolia,Echium plantagineum, Elatine triandra var, pedicellata, Euphorbiaheterophylla, Fallopia convolvulus, Fimbristylis miliacea, Galeopsistetrahit, Galium spurium, Helianthus annuus, Iva xanthifolia, Ixophorusunisetus, Ipomoea indica, Ipomoea purpurea, Ipomoea sepiaria, Ipomoeaaquatic, Ipomoea triloba, Lactuca serriola, Limnocharis flava,Limnophila erecta, Limnophila sessiliflora, Lindernia dubia, Linderniadubia var, major, Lindernia micrantha, Lindernia procumbens,Mesembryanthemum crystallinum, Monochoria korsakowii, Monochoriavaginalis, Neslia paniculata, Papaver rhoeas, Parthenium hysterophorus,Pentzia suffruticosa, Phalaris minor, Raphanus raphanistrum, Raphanussativus, Rapistrum rugosum, Rotala indica var, uliginosa, Sagittariaguyanensis, Sagittaria montevidensis, Sagittaria pygmaea, Salsolaiberica, Scirpus juncoides var, ohwianus, Scirpus mucronatus, Setarialutescens, Sida spinosa, Sinapis arvensis, Sisymbrium orientale,Sisymbrium thellungii, Solanum ptycanthum, Sonchus aspen, Sonchusoleraceus, Sorghum bicolor, Stellaria media, Thlaspi arvense, Xanthiumstrumarium, Arctotheca calendula, Conyza sumatrensis, Crassocephalumcrepidiodes, Cuphea carthagenenis, Epilobium adenocaulon, Erigeronphiladelphicus, Landoltia punctata, Lepidium virginicum, Monochoriakorsakowii, Solanum americanum, Solanum nigrum, Vulpia bromoides,Youngia japonica, Hydrilla verticillata, Carduus nutans, Carduuspycnocephalus, Centaurea solstitialis, Cirsium arvense, Commelinadiffusa, Convolvulus arvensis, Daucus carota, Digitaria ischaemum,Echinochloa crus-pavonis, Fimbristylis miliacea, Galeopsis tetrahit,Galium spurium, Limnophila erecta, Matricaria perforate, Papaver rhoeas,Ranunculus acris, Soliva sessilis, Sphenoclea zeylanica, Stellariamedia, Nassella trichotoma, Stipa neesiana, Agrostis stolonifera,Polygonum aviculare, Alopecurus japonicus, Beckmannia syzigachne, Bromustectorum, Chloris inflate, Echinochloa erecta, Portulaca oleracea, andSenecio vulgaris. It is believed that all plants contain a glutaminesynthetase (GS) gene in their genome, the sequence of which can beisolated and polynucleotides made according to the methods of thepresent invention that are useful for regulation, suppressing ordelaying the expression of the target GS gene in the plants and thegrowth or development of the treated plants.

Some cultivated plants may also be weedy plants when they occur inunwanted environments. For example, corn plants growing in a soybeanfield. Transgenic crops with one or more herbicide tolerances will needspecialized methods of management to control weeds and volunteer cropplants. The present invention enables the targeting of a transgene forherbicide tolerance to permit the treated plants to become sensitive tothe herbicide. For example, transgene GS DNA sequences in transgenicevents that include but are not limited to DP-004114-3, DAS-44406-6,DAS-68416-4, T304-40XGHB119, LLRICE601, TC-6275, LLCotton25, MS1 &RF1/RF2, Topas 19/2, Line 1507, MS6, GU262, A5547-127, T-120-7, W62,W98, A2704-12, A2704-21, A5547-35, and B16.

A “trigger” or “trigger polynucleotide” of the present invention is apolynucleotide molecule that is homologous or complementary to a targetgene polynucleotide. The trigger polynucleotide molecules modulateexpression of the target gene when topically applied to a plant surfacewith a transfer agent, whereby a plant treated with said composition hasits growth or development or reproductive ability regulated, suppressedor delayed or said plant is more sensitive to a GS inhibitor herbicideas a result of said polynucleotide containing composition relative to aplant not treated with a composition containing the trigger molecule.Trigger polynucleotides disclosed herein are generally described inrelation to the target gene sequence and maybe used in the sense(homologous) or antisense (complementary) orientation as single strandedmolecules or comprise both strands as double stranded molecules ornucleotide variants and modified nucleotides thereof depending on thevarious regions of a gene being targeted.

It is contemplated that the composition of the present invention willcontain multiple polynucleotides and herbicides that include but notlimited to GS gene trigger polynucleotides and a GS inhibitor herbicideand anyone or more additional herbicide target gene triggerpolynucleotides and the related herbicides and anyone or more additionalessential gene trigger polynucleotides. Essential genes are genes in aplant that provide key enzymes or other proteins, for example, abiosynthetic enzyme, metabolizing enzyme, receptor, signal transductionprotein, structural gene product, transcription factor, or transportprotein; or regulating RNAs, such as, microRNAs, that are essential tothe growth or survival of the organism or cell or involved in the normalgrowth and development of the plant (Meinke, et al., Trends Plant Sci.2008 September; 13(9):483-91). The suppression of an essential geneenhances the effect of a herbicide that affects the function of a geneproduct different than the suppressed essential gene. The compositionsof the present invention can include various trigger polynucleotidesthat modulate the expression of an essential gene other than a GS gene.

Herbicides for which transgenes for plant tolerance have beendemonstrated and the method of the present invention can be applied,include but are not limited to: auxin-like herbicides, glyphosate,glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon,dicamba, cyclohezanedione, protoporphyrionogen oxidase inhibitors,4-hydroxyphenyl-pyruvate-dioxygenase inhibitors herbicides. For example,transgenes and their polynucleotide molecules that encode proteinsinvolved in herbicide tolerance are known in the art, and include, butare not limited to an 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS), for example, as more fully described in U.S. Pat. No. 7,807,791(SEQ ID NO: 5); U.S. Pat. Nos. 6,248,876 B1; 5,627,061; 5,804,425;5,633,435; 5,145,783; 4,971,908; 5,3372,910; 5,188,642; 4,940,835;5,866,775; 6,225,114 B1; 6,130,366; 5,3370,667; 4,535,060; 4,769,061;5,633,448; 5,510,471; U.S. Pat. No. Re. 36,449; U.S. Pat. Nos. RE 37,287E; and 5,491,288; tolerance to sulfonylurea and/or imidazolinone, forexample, as described more fully in U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,7337,180; 5,304,732; 4,761,373; 5,3337,107;5,928,937; and 5,378,824; and international publication WO 96/33270;tolerance to hydroxyphenylpyruvatedioxygenases inhibiting herbicides inplants are described in U.S. Pat. Nos. 6,245,968 B1; 6,268,549; and6,069,115; US Pat. Pub. 20110191897 and U.S. Pat. No. 7,3372,379 SEQ IDNO: 3; U.S. Pat. No. 7,935,869; U.S. Pat. No. 7,304,209, SEQ ID NOs: 1,3,5 and 15; aryloxyalkanoate dioxygenase polynucleotides, which confertolerance to 2,4-D and other phenoxy auxin herbicides as well as toaryloxyphenoxypropionate herbicides as described, for example, inWO2005/107437; U.S. Pat. No. 7,838,733 SEQ ID NO: 5;) anddicamba-tolerance polynucleotides as described, for example, in Hermanet al. (2005) 1 Biol. Chem. 280: 24759-24767. Other examples ofherbicide-tolerance traits include those conferred by polynucleotidesencoding an exogenous phosphinothricin acetyltransferase, as describedin U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,3378; 5,874,265;5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616; and 5,879,903.Plants containing an exogenous phosphinothricin acetyltransferase canexhibit improved tolerance to glufosinate herbicides, which inhibit theenzyme glutamine synthetase. Additionally, herbicide-tolerancepolynucleotides include those conferred by polynucleotides conferringaltered protoporphyrinogen oxidase (protox) activity, as described inU.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373; and WO01/12825. Plants containing such polynucleotides can exhibit improvedtolerance to any of a variety of herbicides which target the protoxenzyme (also referred to as protox inhibitors). Polynucleotides encodinga glyphosate oxidoreductase and a glyphosate-N-acetyl transferase (GOXdescribed in U.S. Pat. No. 5,463,175 and GAT described in U.S. Patentpublication 20030083480, dicamba monooxygenase U.S. Patent publication20030135879, all of which are incorporated herein by reference); apolynucleotide molecule encoding bromoxynil nitrilase (Bxn described inU.S. Pat. No. 4,810,648 for Bromoxynil tolerance, which is incorporatedherein by reference); a polynucleotide molecule encoding phytoenedesaturase (crtI) described in Misawa et al, (1993) Plant J. 4:833-840and Misawa et al, (1994) Plant J. 6:481-489 for norflurazon tolerance; apolynucleotide molecule encoding acetohydroxyacid synthase (AHAS, akaALS) described in Sathasiivan et al. (1990) Nucl. Acids Res.18:3378-2193 for tolerance to sulfonylurea herbicides; and the bar genedescribed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for glufosinateand bialaphos tolerance. The transgenic coding regions and regulatoryelements of the herbicide tolerance genes are targets in whichpolynucleotide triggers and herbicides can be included in thecomposition of the present invention.

The compositions include a component that is a GS inhibitor herbicide,which include members of the Phosphinic acids herbicide group such asglufosinate-ammonium and bialaphos.

Numerous herbicides with similar or different modes of action (hereinreferred to as co-herbicides) are available that can be added to thecomposition of the present invention, for example, members of theherbicide families that include but are not limited to amide herbicides,aromatic acid herbicides, arsenical herbicides, benzothiazoleherbicides, benzoylcyclohexanedione herbicides, benzofuranylalkylsulfonate herbicides, carbamate herbicides, cyclohexene oximeherbicides, cyclopropylisoxazole herbicides, dicarboximide herbicides,dinitroaniline herbicides, dinitrophenol herbicides, diphenyl etherherbicides, dithiocarbamate herbicides, halogenated aliphaticherbicides, imidazolinone herbicides, inorganic herbicides, nitrileherbicides, organophosphorus herbicides, oxadiazolone herbicides,oxazole herbicides, phenoxy herbicides, phenylenediamine herbicides,pyrazole herbicides, pyridazine herbicides, pyridazinone herbicides,pyridine herbicides, pyrimidinediamine herbicides,pyrimidinyloxybenzylamine herbicides, quaternary ammonium herbicides,thiocarbamate herbicides, thiocarbonate herbicides, thiourea herbicides,triazine herbicides, triazinone herbicides, triazole herbicides,triazolone herbicides, triazolopyrimidine herbicides, uracil herbicides,and urea herbicides. In particular, the rates of use of the addedherbicides can be reduced in compositions comprising the polynucleotidesof the invention. Use rate reductions of the additional added herbicidescan be 10-25 percent, 26-50 percent, 51-75 percent or more can beachieved that enhance the activity of the polynucleotides and herbicidecomposition and is contemplated as an aspect of the invention.Representative co-herbicides of the families include but are not limitedto acetochlor, acifluorfen, acifluorfen-sodium, aclonifen, acrolein,alachlor, alloxydim, allyl alcohol, ametryn, amicarbazone,amidosulfuron, aminopyralid, amitrole, ammonium sulfamate, anilofos,asulam, atraton, atrazine, azimsulfuron, BCPC, beflubutamid, benazolin,benfluralin, benfuresate, bensulfuron, bensulfuron-methyl, bensulide,bentazone, benzfendizone, benzobicyclon, benzofenap, bifenox, bilanafos,bispyribac, bispyribac-sodium, borax, bromacil, bromobutide, bromoxynil,butachlor, butafenacil, butamifos, butralin, butroxydim, butylate,cacodylic acid, calcium chlorate, cafenstrole, carbetamide,carfentrazone, carfentrazone-ethyl, CDEA, CEPC, chlorflurenol,chlorflurenol-methyl, chloridazon, chlorimuron, chlorimuron-ethyl,chloroacetic acid, chlorotoluron, chlorpropham, chlorsulfuron,chlorthal, chlorthal-dimethyl, cinidon-ethyl, cinmethylin, cinosulfuron,cisanilide, clethodim, clodinafop, clodinafop-propargyl, clomazone,clomeprop, clopyralid, cloransulam, cloransulam-methyl, CMA, 4-CPB,CPMF, 4-CPP, CPPC, cresol, cumyluron, cyanamide, cyanazine, cycloate,cyclosulfamuron, cycloxydim, cyhalofop, cyhalofop-butyl, 2,4-D, 3,4-DA,daimuron, dalapon, dazomet, 2,4-DB, 3,4-DB, 2,4-DEB, desmedipham,dicamba, dichlobenil, ortho-dichlorobenzene, para-dichlorobenzene,dichlorprop, dichlorprop-P, diclofop, diclofop-methyl, diclosulam,difenzoquat, difenzoquat metilsulfate, diflufenican, diflufenzopyr,dimefuron, dimepiperate, dimethachlor, dimethametryn, dimethenamid,dimethenamid-P, dimethipin, dimethylarsinic acid, dinitramine, dinoterb,diphenamid, diquat, diquat dibromide, dithiopyr, diuron, DNOC, 3,4-DP,DSMA, EBEP, endothal, EPTC, esprocarb, ethalfluralin, ethametsulfuron,ethametsulfuron-methyl, ethofumesate, ethoxyfen, ethoxysulfuron,etobenzanid, fenoxaprop-P, fenoxaprop-P-ethyl, fentrazamide, ferroussulfate, flamprop-M, flazasulfuron, florasulam, fluazifop,fluazifop-butyl, fluazifop-P, fluazifop-P-butyl, flucarbazone,flucarbazone-sodium, flucetosulfuron, fluchloralin, flufenacet,flufenpyr, flufenpyr-ethyl, flumetsulam, flumiclorac,flumiclorac-pentyl, flumioxazin, fluometuron, fluoroglycofen,fluoroglycofen-ethyl, flupropanate, flupyrsulfuron,flupyrsulfuron-methyl-sodium, flurenol, fluridone, fluorochloridone,fluoroxypyr, flurtamone, fluthiacet, fluthiacet-methyl, fomesafen,foramsulfuron, fosamine, glufosinate, glufosinate-ammonium, glyphosate,halosulfuron, halosulfuron-methyl, haloxyfop, haloxyfop-P, HC-252,hexazinone, imazamethabenz, imazamethabenz-methyl, imazamox, imazapic,imazapyr, imazaquin, imazethapyr, imazosulfuron, indanofan, iodomethane,iodosulfuron, iodosulfuron-methyl-sodium, ioxynil, isoproturon, isouron,isoxaben, isoxachlortole, isoxaflutole, karbutilate, lactofen, lenacil,linuron, MAA, MAMA, MCPA, MCPA-thioethyl, MCPB, mecoprop, mecoprop-P,mefenacet, mefluidide, mesosulfuron, mesosulfuron-methyl, mesotrione,metam, metamifop, metamitron, metazachlor, methabenzthiazuron,methylarsonic acid, methyldymron, methyl isothiocyanate, metobenzuron,metolachlor, S-metolachlor, metosulam, metoxuron, metribuzin,metsulfuron, metsulfuron-methyl, MK-66, molinate, monolinuron, MSMA,naproanilide, napropamide, naptalam, neburon, nicosulfuron, nonanoicacid, norflurazon, oleic acid (fatty acids), orbencarb, orthosulfamuron,oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefone,oxyfluorfen, paraquat, paraquat dichloride, pebulate, pendimethalin,penoxsulam, pentachlorophenol, pentanochlor, pentoxazone, pethoxamid,petrolium oils, phenmedipham, phenmedipham-ethyl, picloram, picolinafen,pinoxaden, piperophos, potassium arsenite, potassium azide,pretilachlor, primisulfuron, primisulfuron-methyl, prodiamine,profluazol, profoxydim, prometon, prometryn, propachlor, propanil,propaquizafop, propazine, propham, propisochlor, propoxycarbazone,propoxycarbazone-sodium, propyzamide, prosulfocarb, prosulfuron,pyraclonil, pyraflufen, pyraflufen-ethyl, pyrazolynate, pyrazosulfuron,pyrazosulfuron-ethyl, pyrazoxyfen, pyribenzoxim, pyributicarb,pyridafol, pyridate, pyriftalid, pyriminobac, pyriminobac-methyl,pyrimisulfan, pyrithiobac, pyrithiobac-sodium, quinclorac, quinmerac,quinoclamine, quizalofop, quizalofop-P, rimsulfuron, sethoxydim,siduron, simazine, simetryn, SMA, sodium arsenite, sodium azide, sodiumchlorate, sulcotrione, sulfentrazone, sulfometuron, sulfometuron-methyl,sulfosate, sulfosulfuron, sulfuric acid, tar oils, 2,3,6-TBA, TCA,TCA-sodium, tebuthiuron, tepraloxydim, terbacil, terbumeton,terbuthylazine, terbutryn, thenylchlor, thiazopyr, thifensulfuron,thifensulfuron-methyl, thiobencarb, tiocarbazil, topramezone,tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron,tribenuron-methyl, tricamba, triclopyr, trietazine, trifloxysulfuron,trifloxysulfuron-sodium, trifluralin, triflusulfuron,triflusulfuron-methyl, trihydroxytriazine, tritosulfuron,[3-[2-chloro-4-fluoro-5-(-methyl-6-trifluoromethyl-2,4-dioxo-,2,3,4-t-etrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]aceticacid ethyl ester (CAS RN 353292-3-6),4-[(4,5-dihydro-3-methoxy-4-methyl-5-oxo)-H-,2,4-triazol-1-ylcarbonyl-sulfamoyl]-5-methylthiophene-3-carboxylicacid (BAY636), BAY747 (CAS RN 33504-84-2), topramezone (CAS RN2063-68-8),4-hydroxy-3-[[2-[(2-methoxyethoxy)methyl]-6-(trifluoro-methyl)-3-pyridi-nyl]carbonyl]-bicyclo[3.2]oct-3-en-2-one(CAS RN 35200-68-5), and4-hydroxy-3-[[2-(3-methoxypropyl)-6-(difluoromethyl)-3-pyridinyl]carbon-yl]-bicyclo[3.2]oct-3-en-2-one.Additionally, including herbicidal compounds of unspecified modes ofaction as described in CN101279950A, CN101279951A, DE10000600A1,DE10116399A1, DE102004054666A1, DE102005014638A1, DE102005014906A1,DE102007012168A1, DE102010042866A1, DE10204951A1, DE10234875A1,DE10234876A1, DE10256353A1, DE10256354A1, DE10256367A1, EP1157991A2,EP1238586A1, EP2147919A1, EP2160098A2, JP03968012B2, JP2001253874A,JP2002080454A, JP2002138075A, JP2002145707A, JP2002220389A,JP2003064059A, JP2003096059A, JP2004051628A, JP2004107228A,JP2005008583A, JP2005239675A, JP2005314407A, JP2006232824A,JP2006282552A, JP2007153847A, JP2007161701A, JP2007182404A,JP2008074840A, JP2008074841A, JP2008133207A, JP2008133218A,JP2008169121A, JP2009067739A, JP2009114128A, JP2009126792A,JP2009137851A, US20060111241A1, US20090036311A1, US20090054240A1,US20090215628A1, US20100099561A1, US20100152443A1, US20110105329A1,US20110201501A1, WO2001055066A2, WO2001056975A1, WO2001056979A1,WO2001090071A2, WO2001090080A1, WO2002002540A1, WO2002028182A1,WO2002040473A1, WO2002044173A2, WO2003000679A2, WO2003006422A1,WO2003013247A1, WO2003016308A1, WO2003020704A1, WO2003022051A1,WO2003022831A1, WO2003022843A1, WO2003029243A2, WO2003037085A1,WO2003037878A1, WO2003045878A2, WO2003050087A2, WO2003051823A1,WO2003051824A1, WO2003051846A2, WO2003076409A1, WO2003087067A1,WO2003090539A1, WO2003091217A1, WO2003093269A2, WO2003104206A2,WO2004002947A1, WO2004002981A2, WO2004011429A1, WO2004029060A1,WO2004035545A2, WO2004035563A1, WO2004035564A1, WO2004037787A1,WO2004067518A1, WO2004067527A1, WO2004077950A1, WO2005000824A1,WO2005007627A1, WO2005040152A1, WO2005047233A1, WO2005047281A1,WO2005061443A2, WO2005061464A1, WO2005068434A1, WO2005070889A1,WO2005089551A1, WO2005095335A1, WO2006006569A1, WO2006024820A1,WO2006029828A1, WO2006029829A1, WO2006037945A1, WO2006050803A1,WO2006090792A1, WO2006123088A2, WO2006125687A1, WO2006125688A1,WO2007003294A1, WO2007026834A1, WO2007071900A1, WO2007077201A1,WO2007077247A1, WO2007096576A1, WO2007119434A1, WO2007134984A1,WO2008009908A1, WO2008029084A1, WO2008059948A1, WO2008071918A1,WO2008074991A1, WO2008084073A1, WO2008100426A2, WO2008102908A1,WO2008152072A2, WO2008152073A2, WO2009000757A1, WO2009005297A2,WO2009035150A2, WO2009063180A1, WO2009068170A2, WO2009068171A2,WO2009086041A1, WO2009090401A2, WO2009090402A2, WO2009115788A1,WO2009116558A1, WO2009152995A1, WO2009158258A1, WO2010012649A1,WO2010012649A1, WO2010026989A1, WO2010034153A1, WO2010049270A1,WO2010049369A1, WO2010049405A1, WO2010049414A1, WO2010063422A1,WO2010069802A1, WO2010078906A2, WO2010078912A1, WO2010104217A1,WO2010108611A1, WO2010112826A3, WO2010116122A3, WO2010119906A1,WO2010130970A1, WO2011003776A2, WO2011035874A1, WO2011065451A1, all ofwhich are incorporated herein by reference.

An agronomic field in need of plant control is treated by application ofthe composition directly to the surface of the growing plants, such asby a spray. For example, the method is applied to control weeds in afield of crop plants by spraying the field with the composition. Thecomposition can be provided as a tank mix, a sequential treatment ofcomponents (generally the polynucleotide containing composition followedby the herbicide), or a simultaneous treatment or mixing of one or moreof the components of the composition from separate containers. Treatmentof the field can occur as often as needed to provide weed control andthe components of the composition can be adjusted to target specificweed species or weed families through utilization of specificpolynucleotides or polynucleotide compositions capable of selectivelytargeting the specific species or plant family to be controlled. Thecomposition can be applied at effective use rates according to the timeof application to the field, for example, preplant, at planting, postplanting, post harvest. GS inhibitor herbicides can be applied to afield at rates of 100 to 500 g ai/ha (active ingredient per hectare) ormore. The polynucleotides of the composition can be applied at rates of1 to 30 grams per acre depending on the number of trigger moleculesneeded for the scope of weeds in the field.

Crop plants in which weed control is needed include but are not limitedto, i) corn, soybean, cotton, canola, sugar beet, alfalfa, sugarcane,rice, and wheat; ii) vegetable plants including, but not limited to,tomato, sweet pepper, hot pepper, melon, watermelon, cucumber, eggplant,cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweetcorn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish,Brussels sprouts, tomatillo, garden beans, dry beans, or okra; iii)culinary plants including, but not limited to, basil, parsley, coffee,or tea; or, iv) fruit plants including but not limited to apple, pear,cherry, peach, plum, apricot, banana, plantain, table grape, wine grape,citrus, avocado, mango, or berry; v) a tree grown for ornamental orcommercial use, including, but not limited to, a fruit or nut tree; or,vi) an ornamental plant (e. g., an ornamental flowering plant or shrubor turf grass). The methods and compositions provided herein can also beapplied to plants produced by a cutting, cloning, or grafting process(i. e., a plant not grown from a seed) include fruit trees and plantsthat include, but are not limited to, citrus, apples, avocados,tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well asvarious ornamental plants.

Pesticidal Mixtures

The polynucleotide compositions may also be used as mixtures withvarious agricultural chemicals and/or insecticides, miticides andfungicides, pesticidal and biopesticidal agents. Examples include butare not limited to azinphos-methyl, acephate, isoxathion, isofenphos,ethion, etrimfos, oxydemeton-methyl, oxydeprofos, quinalphos,chlorpyrifos, chlorpyrifos-methyl, chlorfenvinphos, cyanophos,dioxabenzofos, dichlorvos, disulfoton, dimethylvinphos, dimethoate,sulprofos, diazinon, thiometon, tetrachlorvinphos, temephos,tebupirimfos, terbufos, naled, vamidothion, pyraclofos, pyridafenthion,pirimiphos-methyl, fenitrothion, fenthion, phenthoate, flupyrazophos,prothiofos, propaphos, profenofos, phoxime, phosalone, phosmet,formothion, phorate, malathion, mecarb am, mesulfenfos, methamidophos,methidathion, parathion, methyl parathion, monocrotophos, trichlorphon,EPN, isazophos, isamidofos, cadusafos, diamidaphos, dichlofenthion,thionazin, fenamiphos, fosthiazate, fosthietan, phosphocarb, DSP,ethoprophos, alanycarb, aldicarb, isoprocarb, ethiofencarb, carbaryl,carbosulfan, xylylcarb, thiodicarb, pirimicarb, fenobucarb,furathiocarb, propoxur, bendiocarb, benfuracarb, methomyl, metolcarb,XMC, carbofuran, aldoxycarb, oxamyl, acrinathrin, allethrin,esfenvalerate, empenthrin, cycloprothrin, cyhalothrin,gamma-cyhalothrin, lambda-cyhalothrin, cyfluthrin, beta-cyfluthrin,cypermethrin, alpha-cypermethrin, zeta-cypermethrin, silafluofen,tetramethrin, tefluthrin, deltamethrin, tralomethrin, bifenthrin,phenothrin, fenvalerate, fenpropathrin, furamethrin, prallethrin,flucythrinate, fluvalinate, flubrocythrinate, permethrin, resmethrin,ethofenprox, cartap, thiocyclam, bensultap, acetamiprid, imidacloprid,clothianidin, dinotefuran, thiacloprid, thiamethoxam, nitenpyram,chlorfluazuron, diflubenzuron, teflubenzuron, triflumuron, novaluron,noviflumuron, bistrifluoron, fluazuron, flucycloxuron, flufenoxuron,hexaflumuron, lufenuron, chromafenozide, tebufenozide, halofenozide,methoxyfenozide, diofenolan, cyromazine, pyriproxyfen, buprofezin,methoprene, hydroprene, kinoprene, triazamate, endosulfan, chlorfenson,chlorobenzilate, dicofol, bromopropylate, acetoprole, fipronil,ethiprole, pyrethrin, rotenone, nicotine sulphate, BT (BacillusThuringiensis) agent, spinosad, abamectin, acequinocyl, amidoflumet,amitraz, etoxazole, chinomethionat, clofentezine, fenbutatin oxide,dienochlor, cyhexatin, spirodiclofen, spiromesifen, tetradifon,tebufenpyrad, binapacryl, bifenazate, pyridaben, pyrimidifen,fenazaquin, fenothiocarb, fenpyroximate, fluacrypyrim, fluazinam,flufenzin, hexythiazox, propargite, benzomate, polynactin complex,milbemectin, lufenuron, mecarbam, methiocarb, mevinphos, halfenprox,azadirachtin, diafenthiuron, indoxacarb, emamectin benzoate, potassiumoleate, sodium oleate, chlorfenapyr, tolfenpyrad, pymetrozine,fenoxycarb, hydramethylnon, hydroxy propyl starch, pyridalyl,flufenerim, flubendiamide, flonicamid, metaflumizole, lepimectin, TPIC,albendazole, oxibendazole, oxfendazole, trichlamide, fensulfothion,fenbendazole, levamisole hydrochloride, morantel tartrate, dazomet,metam-sodium, triadimefon, hexaconazole, propiconazole, ipconazole,prochloraz, triflumizole, tebuconazole, epoxiconazole, difenoconazole,flusilazole, triadimenol, cyproconazole, metconazole, fluquinconazole,bitertanol, tetraconazole, triticonazole, flutriafol, penconazole,diniconazole, fenbuconazole, bromuconazole, imibenconazole,simeconazole, myclobutanil, hymexazole, imazalil, furametpyr,thifluzamide, etridiazole, oxpoconazole, oxpoconazole fumarate,pefurazoate, prothioconazole, pyrifenox, fenarimol, nuarimol,bupirimate, mepanipyrim, cyprodinil, pyrimethanil, metalaxyl, mefenoxam,oxadixyl, benalaxyl, thiophanate, thiophanate-methyl, benomyl,carbendazim, fuberidazole, thiabendazole, manzeb, propineb, zineb,metiram, maneb, ziram, thiuram, chlorothalonil, ethaboxam, oxycarboxin,carboxin, flutolanil, silthiofam, mepronil, dimethomorph, fenpropidin,fenpropimorph, spiroxamine, tridemorph, dodemorph, flumorph,azoxystrobin, kresoxim-methyl, metominostrobin, orysastrobin,fluoxastrobin, trifloxystrobin, dimoxystrobin, pyraclostrobin,picoxystrobin, iprodione, procymidone, vinclozolin, chlozolinate,flusulfamide, dazomet, methyl isothiocyanate, chloropicrin,methasulfocarb, hydroxyisoxazole, potassium hydroxyisoxazole,echlomezol, D-D, carbam, basic copper chloride, basic copper sulfate,copper nonylphenolsulfonate, oxine copper, DBEDC, anhydrous coppersulfate, copper sulfate pentahydrate, cupric hydroxide, inorganicsulfur, wettable sulfur, lime sulfur, zinc sulfate, fentin, sodiumhydrogen carbonate, potassium hydrogen carbonate, sodium hypochlorite,silver, edifenphos, tolclofos-methyl, fosetyl, iprobenfos, dinocap,pyrazophos, carpropamid, fthalide, tricyclazole, pyroquilon, diclocymet,fenoxanil, kasugamycin, validamycin, polyoxins, blasticiden S,oxytetracycline, mildiomycin, streptomycin, rape seed oil, machine oil,benthiavalicarbisopropyl, iprovalicarb, propamocarb, diethofencarb,fluoroimide, fludioxanil, fenpiclonil, quinoxyfen, oxolinic acid,chlorothalonil, captan, folpet, probenazole, acibenzolar-S-methyl,tiadinil, cyflufenamid, fenhexamid, diflumetorim, metrafenone,picobenzamide, proquinazid, famoxadone, cyazofamid, fenamidone,zoxamide, boscalid, cymoxanil, dithianon, fluazinam, dichlofluanide,triforine, isoprothiolane, ferimzone, diclomezine, tecloftalam,pencycuron, chinomethionat, iminoctadine acetate, iminoctadinealbesilate, ambam, polycarbamate, thiadiazine, chloroneb, nickeldimethyldithiocarbamate, guazatine, dodecylguanidine-acetate,quintozene, tolylfluanid, anilazine, nitrothalisopropyl, fenitropan,dimethirimol, benthiazole, harpin protein, flumetover, mandipropamideand penthiopyrad.

Polynucleotides

As used herein, the term “DNA”, “DNA molecule”, “DNA polynucleotidemolecule” refers to a single-stranded DNA (ssDNA) or double-stranded DNA(dsDNA) molecule of genomic or synthetic origin, such as, a polymer ofdeoxyribonucleotide bases or a DNA polynucleotide molecule. As usedherein, the term “DNA sequence”, “DNA nucleotide sequence” or “DNApolynucleotide sequence” refers to the nucleotide sequence of a DNAmolecule. As used herein, the term “RNA”, “RNA molecule”, “RNApolynucleotide molecule” refers to a single-stranded RNA (ssRNA) ordouble-stranded RNA (dsRNA) molecule of genomic or synthetic origin,such as, a polymer of ribonucleotide bases that comprise single ordouble stranded regions. Unless otherwise stated, nucleotide sequencesin the text of this specification are given, when read from left toright, in the 5′ to 3′ direction. The nomenclature used herein is thatrequired by Title 37 of the United States Code of Federal Regulations§1.822 and set forth in the tables in WIPO Standard ST.25 (1998),Appendix 2, Tables 1 and 3.

As used herein, “polynucleotide” refers to a DNA or RNA moleculecontaining multiple nucleotides and generally refers both to“oligonucleotides” (a polynucleotide molecule of typically 50 or fewernucleotides in length) and polynucleotides of 51 or more nucleotides.Embodiments of this invention include compositions includingoligonucleotides having a length of 18-25 nucleotides (18-mers, 19-mers,20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers) for example,oligonucleotides SEQ ID NOs:1444-2045 or fragments thereof, ormedium-length polynucleotides having a length of 26 or more nucleotides(polynucleotides of 26, 27, 28, 29, 30, 337, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about90, about 95, about 100, about 110, about 120, about 130, about 140,about 150, about 160, about 170, about 180, about 190, about 200, about210, about 220, about 230, about 240, about 250, about 260, about 270,about 280, about 290, or about 300 nucleotides), for example,oligonucleotides of SEQ ID NOs: 60-1443 or fragments thereof or longpolynucleotides having a length greater than about 300 nucleotides (forexample, polynucleotides of between about 300 to about 400 nucleotides,between about 400 to about 500 nucleotides, between about 500 to about600 nucleotides, between about 600 to about 700 nucleotides, betweenabout 700 to about 800 nucleotides, between about 800 to about 900nucleotides, between about 900 to about 1000 nucleotides, between about300 to about 500 nucleotides, between about 300 to about 600nucleotides, between about 300 to about 700 nucleotides, between about300 to about 800 nucleotides, between about 300 to about 900nucleotides, or about 1000 nucleotides in length, or even greater thanabout 1000 nucleotides in length, for example up to the entire length ofa target gene including coding or non-coding or both coding andnon-coding portions of the target gene), for example, polynucleotides ofTable 1 (SEQ ID NOs: 1-59), wherein the selected polynucleotides orfragments thereof are homologous or complementary to SEQ ID NOs: 1-59,suppresses, represses or otherwise delays the expression of the targetGS gene. A target gene comprises any polynucleotide molecule in a plantcell or fragment thereof for which the modulation of the expression ofthe target gene is provided by the methods and compositions of thepresent invention. Where a polynucleotide is double-stranded, its lengthcan be similarly described in terms of base pairs. Oligonucleotides andpolynucleotides of the present invention can be made that areessentially identical or essentially complementary to adjacent geneticelements of a gene, for example, spanning the junction region of anintron and exon, the junction region of a promoter and a transcribedregion, the junction region of a 5′ leader and a coding sequence, thejunction of a 3′ untranslated region and a coding sequence.

Polynucleotide compositions used in the various embodiments of thisinvention include compositions including oligonucleotides orpolynucleotides or a mixture of both, including RNA or DNA or RNA/DNAhybrids or chemically modified oligonucleotides or polynucleotides or amixture thereof. In some embodiments, the polynucleotide may be acombination of ribonucleotides and deoxyribonucleotides, for example,synthetic polynucleotides consisting mainly of ribonucleotides but withone or more terminal deoxyribonucleotides or synthetic polynucleotidesconsisting mainly of deoxyribonucleotides but with one or more terminaldideoxyribonucleotides. In some embodiments, the polynucleotide includesnon-canonical nucleotides such as inosine, thiouridine, orpseudouridine. In some embodiments, the polynucleotide includeschemically modified nucleotides. Examples of chemically modifiedoligonucleotides or polynucleotides are well known in the art; see, forexample, US Patent Publication 20110171287, US Patent Publication20110171176, and US Patent Publication 20110152353, US PatentPublication, 20110152346, US Patent Publication 20110160082, hereinincorporated by reference. For example, including but not limited to thenaturally occurring phosphodiester backbone of an oligonucleotide orpolynucleotide can be partially or completely modified withphosphorothioate, phosphorodithioate, or methylphosphonateinternucleotide linkage modifications, modified nucleoside bases ormodified sugars can be used in oligonucleotide or polynucleotidesynthesis, and oligonucleotides or polynucleotides can be labeled with afluorescent moiety (for example, fluorescein or rhodamine) or otherlabel (for example, biotin).

The polynucleotides can be single- or double-stranded RNA or single- ordouble-stranded DNA or double-stranded DNA/RNA hybrids or modifiedanalogues thereof, and can be of oligonucleotide lengths or longer. Inmore specific embodiments of the invention the polynucleotides thatprovide single-stranded RNA in the plant cell are selected from thegroup consisting of (a) a single-stranded RNA molecule (ssRNA), (b) asingle-stranded RNA molecule that self-hybridizes to form adouble-stranded RNA molecule, (c) a double-stranded RNA molecule(dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) asingle-stranded DNA molecule that self-hybridizes to form adouble-stranded DNA molecule, and (f) a single-stranded DNA moleculeincluding a modified Pol III gene that is transcribed to an RNAmolecule, (g) a double-stranded DNA molecule (dsDNA), (h) adouble-stranded DNA molecule including a modified Pol III gene that istranscribed to an RNA molecule, (i) a double-stranded, hybridizedRNA/DNA molecule, or combinations thereof. In some embodiments thesepolynucleotides include chemically modified nucleotides or non-canonicalnucleotides. In some embodiments, the oligonucleotides may beblunt-ended or may comprise a 3′ overhang of from 1-5 nucleotides of atleast one or both of the strands. Other configurations of theoligonucleotide are known in the field and are contemplated herein. Inembodiments of the method the polynucleotides include double-strandedDNA formed by intramolecular hybridization, double-stranded DNA formedby intermolecular hybridization, double-stranded RNA formed byintramolecular hybridization, or double-stranded RNA formed byintermolecular hybridization. In one embodiment the polynucleotidesinclude single-stranded DNA or single-stranded RNA that self-hybridizesto form a hairpin structure having an at least partially double-strandedstructure including at least one segment that will hybridize to RNAtranscribed from the gene targeted for suppression. Not intending to bebound by any mechanism, it is believed that such polynucleotides are orwill produce single-stranded RNA with at least one segment that willhybridize to RNA transcribed from the gene targeted for suppression. Incertain other embodiments the polynucleotides further includes apromoter, generally a promoter functional in a plant, for example, a polII promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.

The term “gene” refers to chromosomal DNA, plasmid DNA, cDNA, intron andexon DNA, artificial DNA polynucleotide, or other DNA that encodes apeptide, polypeptide, protein, or RNA transcript molecule, and thegenetic elements flanking the coding sequence that are involved in theregulation of expression, such as, promoter regions, 5′ leader regions,3′ untranslated regions. Any of the components of the gene are potentialtargets for the oligonucleotides and polynucleotides of the presentinvention.

The polynucleotide molecules of the present invention are designed tomodulate expression by inducing regulation or suppression of anendogenous GS gene in a plant and are designed to have a nucleotidesequence essentially identical or essentially complementary to thenucleotide sequence of an endogenous GS gene of a plant or to thesequence of RNA transcribed from an endogenous GS gene of a plant,including a transgene in a plant that provides for a herbicide resistantGS enzyme, which can be coding sequence or non-coding sequence.Effective molecules that modulate expression are referred to as “atrigger molecule, or trigger polynucleotide”. By “essentially identical”or “essentially complementary” is meant that the trigger polynucleotides(or at least one strand of a double-stranded polynucleotide or portionthereof, or a portion of a single strand polynucleotide) are designed tohybridize to the endogenous gene noncoding sequence or to RNAtranscribed (known as messenger RNA or an RNA transcript) from theendogenous gene to effect regulation or suppression of expression of theendogenous gene. Trigger molecules are identified by “tiling” the genetargets with partially overlapping probes or non-overlapping probes ofantisense or sense polynucleotides that are essentially identical oressentially complementary to the nucleotide sequence of an endogenousgene. Multiple target sequences can be aligned and sequence regions withhomology in common, according to the methods of the present invention,are identified as potential trigger molecules for the multiple targets.Multiple trigger molecules of various lengths, for example 18-25nucleotides, 26-50 nucleotides, 51-100 nucleotides, 101-200 nucleotides,201-300 nucleotides or more can be pooled into a few treatments in orderto investigate polynucleotide molecules that cover a portion of a genesequence (for example, a portion of a coding versus a portion of anoncoding region, or a 5′ versus a 3′ portion of a gene) or an entiregene sequence including coding and noncoding regions of a target gene.Polynucleotide molecules of the pooled trigger molecules can be dividedinto smaller pools or single molecules in order to identify triggermolecules that provide the desired effect.

The target gene RNA and DNA polynucleotide molecules (Table 1, SEQ IDNOs: 1-59) are sequenced by any number of available methods andequipment. Some of the sequencing technologies are availablecommercially, such as the sequencing-by-hybridization platform fromAffymetrix Inc. (Sunnyvale, Calif.) and the sequencing-by-synthesisplatforms from 454 Life Sciences (Bradford, Conn.), Illumina/Solexa(Hayward, Calif.) and Helicos Biosciences (Cambridge, Mass.), and thesequencing-by-ligation platform from Applied Biosystems (Foster City,Calif.), as described below. In addition to the single moleculesequencing performed using sequencing-by-synthesis of HelicosBiosciences, other single molecule sequencing technologies areencompassed by the method of the invention and include the SMRT™technology of Pacific Biosciences, the Ion Torrent™ technology, andnanopore sequencing being developed for example, by Oxford NanoporeTechnologies. A GS target gene comprising DNA or RNA can be isolatedusing primers or probes essentially complementary or essentiallyhomologous to SEQ ID NOs: 1-59 or a fragment thereof. A polymerase chainreaction (PCR) gene fragment can be produced using primers essentiallycomplementary or essentially homologous to SEQ ID NOs: 1-59 or afragment thereof that is useful to isolate a GS gene from a plantgenome. SEQ ID NOs: 1-59 or fragments thereof can be used in varioussequence capture technologies to isolate additional target genesequences, for example, including but not limited to Roche NimbleGen®(Madison, Wis.) and Streptavdin-coupled Dynabeads® (Life Technologies,Grand Island, N.Y.) and US20110015084, herein incorporated by referencein its entirety.

Embodiments of functional single-stranded polynucleotides have sequencecomplementarity that need not be 100 percent, but is at least sufficientto permit hybridization to RNA transcribed from the target gene or DNAof the target gene to form a duplex to permit a gene silencingmechanism. Thus, in embodiments, a polynucleotide fragment is designedto be essentially identical to, or essentially complementary to, asequence of 18 or more contiguous nucleotides in either the target GSgene sequence or messenger RNA transcribed from the target gene. By“essentially identical” is meant having 100 percent sequence identity orat least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99 percent sequence identity when compared to the sequence of18 or more contiguous nucleotides in either the target gene or RNAtranscribed from the target gene; by “essentially complementary” ismeant having 100 percent sequence complementarity or at least about 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99percent sequence complementarity when compared to the sequence of 18 ormore contiguous nucleotides in either the target gene or RNA transcribedfrom the target gene. In some embodiments of this inventionpolynucleotide molecules are designed to have 100 percent sequenceidentity with or complementarity to one allele or one family member of agiven target gene (coding or non-coding sequence of a gene for of thepresent invention); in other embodiments the polynucleotide moleculesare designed to have 100 percent sequence identity with orcomplementarity to multiple alleles or family members of a given targetgene.

In certain embodiments, the polynucleotides used in the compositionsthat are essentially identical or essentially complementary to thetarget gene or transcript will comprise the predominant nucleic acid inthe composition. Thus in certain embodiments, the polynucleotides thatare essentially identical or essentially complementary to the targetgene or transcript will comprise at least about 50%, 75%, 95%, 98% or100% of the nucleic acids provided in the composition by either mass ormolar concentration. However, in certain embodiments, thepolynucleotides that are essentially identical or essentiallycomplementary to the target gene or transcript can comprise at leastabout 1% to about 50%, about 10% to about 50%, about 20% to about 50%,or about 30% to about 50% of the nucleic acids provided in thecomposition by either mass or molar concentration. Also provided arecompositions where the polynucleotides that are essentially identical oressentially complementary to the target gene or transcript can compriseat least about 1% to 100%, about 10% to 100%, about 20% to about 100%,about 30% to about 50%, or about 50% to a 100% of the nucleic acidsprovided in the composition by either mass or molar concentration.

“Identity” refers to the degree of similarity between two polynucleicacid or protein sequences. An alignment of the two sequences isperformed by a suitable computer program. A widely used and acceptedcomputer program for performing sequence alignments is CLUSTALW v1.6(Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number ofmatching bases or amino acids is divided by the total number of bases oramino acids, and multiplied by 100 to obtain a percent identity. Forexample, if two 580 base pair sequences had 145 matched bases, theywould be 25 percent identical. If the two compared sequences are ofdifferent lengths, the number of matches is divided by the shorter ofthe two lengths. For example, if there are 100 matched amino acidsbetween a 200 and a 400 amino acid protein, they are 50 percentidentical with respect to the shorter sequence. If the shorter sequenceis less than 150 bases or 50 amino acids in length, the number ofmatches are divided by 150 (for nucleic acid bases) or 50 (for aminoacids), and multiplied by 100 to obtain a percent identity.

Trigger molecules for specific gene family members can be identifiedfrom coding and/or non-coding sequences of gene families of a plant ormultiple plants, by aligning and selecting 200-300 polynucleotidefragments from the least homologous regions amongst the alignedsequences and evaluated using topically applied polynucleotides (assense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine theirrelative effectiveness in inducing the herbicidal phenotype. Theeffective segments are further subdivided into 50-60 polynucleotidefragments, prioritized by least homology, and reevaluated usingtopically applied polynucleotides. The effective 50-60 polynucleotidefragments are subdivided into 19-30 polynucleotide fragments,prioritized by least homology, and again evaluated for induction of theyield/quality phenotype. Once relative effectiveness is determined, thefragments are utilized singly, or again evaluated in combination withone or more other fragments to determine the trigger composition ormixture of trigger polynucleotides for providing the yield/qualityphenotype.

Trigger molecules for broad activity can be identified from codingand/or non-coding sequences of gene families of a plant or multipleplants, by aligning and selecting 200-300 polynucleotide fragments fromthe most homologous regions amongst the aligned sequences and evaluatedusing topically applied polynucleotides (as sense or anti-sense ssDNA orssRNA, dsRNA, or dsDNA) to determine their relative effectiveness ininducing the yield/quality phenotype. The effective segments aresubdivided into 50-60 polynucleotide fragments, prioritized by mosthomology, and reevaluated using topically applied polynucleotides. Theeffective 50-60 polynucleotide fragments are subdivided into 19-30polynucleotide fragments, prioritized by most homology, and againevaluated for induction of the yield/quality phenotype. Once relativeeffectiveness is determined, the fragments may be utilized singly, or incombination with one or more other fragments to determine the triggercomposition or mixture of trigger polynucleotides for providing theyield/quality phenotype.

Methods of making polynucleotides are well known in the art. Chemicalsynthesis, in vivo synthesis and in vitro synthesis methods andcompositions are known in the art and include various viral elements,microbial cells, modified polymerases, and modified nucleotides.Commercial preparation of oligonucleotides often provides twodeoxyribonucleotides on the 3′ end of the sense strand. Longpolynucleotide molecules can be synthesized from commercially availablekits, for example, kits from Applied Biosystems/Ambion (Austin, Tex.)have DNA ligated on the 5′ end in a microbial expression cassette thatincludes a bacterial T7 polymerase promoter that makes RNA strands thatcan be assembled into a dsRNA and kits provided by various manufacturersthat include T7 RiboMax Express (Promega, Madison, Wis.), AmpliScribeT7-Flash (Epicentre, Madison, Wis.), and TranscriptAid T7 High Yield(Fermentas, Glen Burnie, Md.). dsRNA molecules can be produced frommicrobial expression cassettes in bacterial cells (Ongvarrasopone et al.ScienceAsia 33:35-39; Yin, Appl. Microbiol. Biotechnol. 84:323-333,2009; Liu et al., BMC Biotechnology 10:85, 2010) that have regulated ordeficient RNase III enzyme activity or the use of various viral vectorsto produce sufficient quantities of dsRNA. In the present invention, GSgene fragments are inserted into the microbial expression cassettes in aposition in which the fragments are express to produce ssRNA or dsRNAuseful in the methods described herein to regulate expression on atarget GS gene. Long polynucleotide molecules can also be assembled frommultiple RNA or DNA fragments. In some embodiments design parameterssuch as Reynolds score (Reynolds et al. Nature Biotechnology 22, 326-330(2004), Tuschl rules (Pei and Tuschl, Nature Methods 3(9): 670-676,2006), i-score (Nucleic Acids Res 35: e123, 2007), i-Score Designer tooland associated algorithms (Nucleic Acids Res 32: 936-948, 2004. BiochemBiophys Res Commun 316: 1050-1058, 2004, Nucleic Acids Res 32: 893-901,2004, Cell Cycle 3: 790-5, 2004, Nat. Biotechnol. 23: 995-1001, 2005,Nucleic Acids Res. 35: e27, 2007, BMC Bioinformatics 7: 520, 2006,Nucleic Acids Res. 35: e123, 2007, Nat. Biotechnol. 22: 326-330, 2004)are known in the art and may be used in selecting polynucleotidesequences effective in gene silencing. In some embodiments the sequenceof a polynucleotide is screened against the genomic DNA of the intendedplant to minimize unintentional silencing of other genes.

The trigger polynucleotide and oligonucleotide molecule compositions ofthis invention are useful in compositions, such as liquids that comprisethe polynucleotide molecules at low concentrations, alone or incombination with other components, for example one or more herbicidemolecules, either in the same solution or in separately applied liquidsthat also provide a transfer agent. While there is no upper limit on theconcentrations and dosages of polynucleotide molecules that can usefulin the methods, lower effective concentrations and dosages willgenerally be sought for efficiency. The concentrations can be adjustedin consideration of the volume of spray or treatment applied to plantleaves or other plant part surfaces, such as flower petals, stems,tubers, fruit, anthers, pollen, or seed. In one embodiment, a usefultreatment for herbaceous plants using 25-mer oligonucleotide moleculesis about 1 nanomole (nmol) of oligonucleotide molecules per plant, forexample, from about 0.05 to 1 nmol per plant. Other embodiments forherbaceous plants include useful ranges of about 0.05 to about 100 nmol,or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol ofpolynucleotides per plant. Very large plants, trees, or vines mayrequire correspondingly larger amounts of polynucleotides. When usinglong dsRNA molecules that can be processed into multipleoligonucleotides, lower concentrations can be used. To illustrateembodiments of the invention, the factor 1×, when applied tooligonucleotide molecules is arbitrarily used to denote a treatment of0.8 nmol of polynucleotide molecule per plant; 10×, 8 nmol ofpolynucleotide molecule per plant; and 100×, 80 nmol of polynucleotidemolecule per plant.

The polynucleotide compositions of this invention are useful incompositions, such as liquids that comprise polynucleotide molecules,alone or in combination with other components either in the same liquidor in separately applied liquids that provide a transfer agent. As usedherein, a transfer agent is an agent that, when combined with apolynucleotide in a composition that is topically applied to a targetplant surface, enables the polynucleotide to enter a plant cell. Incertain embodiments, a transfer agent is an agent that conditions thesurface of plant tissue, e. g., leaves, stems, roots, flowers, orfruits, to permeation by the polynucleotide molecules into plant cells.The transfer of polynucleotides into plant cells can be facilitated bythe prior or contemporaneous application of apolynucleotide-transferring agent to the plant tissue. In someembodiments the transferring agent is applied subsequent to theapplication of the polynucleotide composition. The polynucleotidetransfer agent enables a pathway for polynucleotides through cuticle waxbarriers, stomata and/or cell wall or membrane barriers into plantcells. Suitable transfer agents to facilitate transfer of thepolynucleotide into a plant cell include agents that increasepermeability of the exterior of the plant or that increase permeabilityof plant cells to oligonucleotides or polynucleotides. Such agents tofacilitate transfer of the composition into a plant cell include achemical agent, or a physical agent, or combinations thereof. Chemicalagents for conditioning or transfer include (a) surfactants, (b) anorganic solvent or an aqueous solution or aqueous mixtures of organicsolvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g)enzymes, or combinations thereof. Embodiments of the method canoptionally include an incubation step, a neutralization step (e.g., toneutralize an acid, base, or oxidizing agent, or to inactivate anenzyme), a rinsing step, or combinations thereof. Embodiments of agentsor treatments for conditioning of a plant to permeation bypolynucleotides include emulsions, reverse emulsions, liposomes, andother micellar-like compositions. Embodiments of agents or treatmentsfor conditioning of a plant to permeation by polynucleotides includecounter-ions or other molecules that are known to associate with nucleicacid molecules, e. g., inorganic ammonium ions, alkyl ammonium ions,lithium ions, polyamines such as spermine, spermidine, or putrescine,and other cations. Organic solvents useful in conditioning a plant topermeation by polynucleotides include DMSO, DMF, pyridine,N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane,polypropylene glycol, other solvents miscible with water or that willdissolve phosphonucleotides in non-aqueous systems (such as is used insynthetic reactions). Naturally derived or synthetic oils with orwithout surfactants or emulsifiers can be used, e. g., plant-sourcedoils, crop oils (such as those listed in the 9th Compendium of HerbicideAdjuvants, publicly available on the worldwide web (internet) atherbicide.adjuvants.com can be used, e. g., paraffinic oils, polyolfatty acid esters, or oils with short-chain molecules modified withamides or polyamines such as polyethyleneimine or N-pyrrolidine.Transfer agents include, but are not limited to, organosiliconepreparations.

In certain embodiments, an organosilicone preparation that iscommercially available as Silwet® L-77 surfactant having CAS Number27306-78-1 and EPA Number: CAL. REG. NO. 5905-50073-AA, and currentlyavailable from Momentive Performance Materials, Albany, N.Y. can be usedto prepare a polynucleotide composition. In certain embodiments where aSilwet L-77 organosilicone preparation is used as a pre-spray treatmentof plant leaves or other plant surfaces, freshly made concentrations inthe range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05,0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious inpreparing a leaf or other plant surface for transfer of polynucleotidemolecules into plant cells from a topical application on the surface. Incertain embodiments of the methods and compositions provided herein, acomposition that comprises a polynucleotide molecule and anorganosilicone preparation comprising Silwet L-77 in the range of about0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01,0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065,0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.5 wt percent) is used or provided.

In certain embodiments, any of the commercially available organosiliconepreparations provided such as the following Breakthru S 321, Breakthru S200 Cat#67674-67-3, Breakthru OE 441 Cat#68937-55-3, Breakthru S 278 Cat#27306-78-1, Breakthru S 243, Breakthru S 233 Cat#134180-76-0, availablefrom manufacturer Evonik Goldschmidt (Germany), Silwet® HS 429, Silwet®HS 312, Silwet® HS 508, Silwet® HS 604 (Momentive Performance Materials,Albany, N.Y.) can be used as transfer agents in a polynucleotidecomposition. In certain embodiments where an organosilicone preparationis used as a pre-spray treatment of plant leaves or other surfaces,freshly made concentrations in the range of about 0.015 to about 2percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025,0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08,0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wtpercent) are efficacious in preparing a leaf or other plant surface fortransfer of polynucleotide molecules into plant cells from a topicalapplication on the surface. In certain embodiments of the methods andcompositions provided herein, a composition that comprises apolynucleotide molecule and an organosilicone preparation in the rangeof about 0.015 to about 2 percent by weight (wt percent) (e. g., about0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06,0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used or provided.

Organosilicone preparations used in the methods and compositionsprovided herein can comprise one or more effective organosiliconecompounds. As used herein, the phrase “effective organosiliconecompound” is used to describe any organosilicone compound that is foundin an organosilicone preparation that enables a polynucleotide to entera plant cell. In certain embodiments, an effective organosiliconecompound can enable a polynucleotide to enter a plant cell in a mannerpermitting a polynucleotide mediated suppression of a target geneexpression in the plant cell. In general, effective organosiliconecompounds include, but are not limited to, compounds that can comprise:i) a trisiloxane head group that is covalently linked to, ii) an alkyllinker including, but not limited to, an n-propyl linker, that iscovalently linked to, iii) a poly glycol chain, that is covalentlylinked to, iv) a terminal group. Trisiloxane head groups of sucheffective organosilicone compounds include, but are not limited to,heptamethyltrisiloxane. Alkyl linkers can include, but are not limitedto, an n-propyl linker. Poly glycol chains include, but are not limitedto, polyethylene glycol or polypropylene glycol. Poly glycol chains cancomprise a mixture that provides an average chain length “n” of about“7.5”. In certain embodiments, the average chain length “n” can varyfrom about 5 to about 14. Terminal groups can include, but are notlimited to, alkyl groups such as a methyl group. Effectiveorganosilicone compounds are believed to include, but are not limitedto, trisiloxane ethoxylate surfactants or polyalkylene oxide modifiedheptamethyl trisiloxane.

In certain embodiments, an organosilicone preparation that comprises anorganosilicone compound comprising a trisiloxane head group is used inthe methods and compositions provided herein. In certain embodiments, anorganosilicone preparation that comprises an organosilicone compoundcomprising a heptamethyltrisiloxane head group is used in the methodsand compositions provided herein. In certain embodiments, anorganosilicone composition that comprises Compound I is used in themethods and compositions provided herein. In certain embodiments, anorganosilicone composition that comprises Compound I is used in themethods and compositions provided herein. In certain embodiments of themethods and compositions provided herein, a composition that comprises apolynucleotide molecule and one or more effective organosiliconecompound in the range of about 0.015 to about 2 percent by weight (wtpercent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04,0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used orprovided.

Compositions of the present invention include but are not limitedcomponents that are one or more polynucleotides essentially identicalto, or essentially complementary to a GS gene sequence (promoter,intron, exon, 5′ untranslated region, 3′ untranslated region), atransfer agent that provides for the polynucleotide to enter a plantcell, a herbicide that complements the action of the polynucleotide, oneor more additional herbicides that further enhance the herbicideactivity of the composition or provide an additional mode of actiondifferent from the complementing herbicide, various salts andstabilizing agents that enhance the utility of the composition as anadmixture of the components of the composition.

Methods include one or more applications of a polynucleotide compositionand one or more applications of a permeability-enhancing agent forconditioning of a plant to permeation by polynucleotides. When the agentfor conditioning to permeation is an organosilicone composition orcompound contained therein, embodiments of the polynucleotide moleculesare double-stranded RNA oligonucleotides, single-stranded RNAoligonucleotides, double-stranded RNA polynucleotides, single-strandedRNA polynucleotides, double-stranded DNA oligonucleotides,single-stranded DNA oligonucleotides, double-stranded DNApolynucleotides, single-stranded DNA polynucleotides, chemicallymodified RNA or DNA oligonucleotides or polynucleotides or mixturesthereof.

Compositions and methods are useful for modulating the expression of anendogenous GS gene (for example, Pest Manag. Sci. 2009; 65: 216-222,GS249 mutants) or transgenic GS gene (for example, U.S. Pat. Nos.7,910,805; 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675;5,561,236; 5,648,477; 5,646,024; 6,177,616; and 5,879,903) in a plantcell. In various embodiments, a GS gene includes coding (protein-codingor translatable) sequence, non-coding (non-translatable) sequence, orboth coding and non-coding sequence. Compositions of the invention caninclude polynucleotides and oligonucleotides designed to target multiplegenes, or multiple segments of one or more genes. The target gene caninclude multiple consecutive segments of a target gene, multiplenon-consecutive segments of a target gene, multiple alleles of a targetgene, or multiple target genes from one or more species.

One aspect is a method for modulating expression of a GS gene in a plantincluding (a) conditioning of a plant to permeation by polynucleotidesand (b) treatment of the plant with the polynucleotide molecules,wherein the polynucleotide molecules include at least one segment of 18or more contiguous nucleotides cloned from or otherwise identified fromthe target GS gene in either anti-sense or sense orientation, wherebythe polynucleotide molecules permeate the interior of the plant andinduce modulation of the target gene. The conditioning andpolynucleotide application can be performed separately or in a singlestep. When the conditioning and polynucleotide application are performedin separate steps, the conditioning can precede or can follow thepolynucleotide application within minutes, hours, or days. In someembodiments more than one conditioning step or more than onepolynucleotide molecule application can be performed on the same plant.In embodiments of the method, the segment can be cloned or identifiedfrom (a) coding (protein-encoding), (b) non-coding (promoter and othergene related molecules), or (c) both coding and non-coding parts of thetarget gene. Non-coding parts include DNA, such as promoter regions orthe RNA transcribed by the DNA that provide RNA regulatory molecules,including but not limited to: introns, 5′ or 3′ untranslated regions,and microRNAs (miRNA), trans-acting siRNAs, natural anti-sense siRNAs,and other small RNAs with regulatory function or RNAs having structuralor enzymatic function including but not limited to: ribozymes, ribosomalRNAs, t-RNAs, aptamers, and riboswitches.

All publications, patents and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

The following examples are included to demonstrate examples of certainpreferred embodiments. It should be appreciated by those of skill in theart that the techniques disclosed in the examples that follow representapproaches the inventors have found function well in the practice of theinvention, and thus can be considered to constitute examples ofpreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments that are disclosed and stillobtain a like or similar result without departing from the spirit andscope.

Examples Example 1 Polynucleotides Related to the GS Gene Sequences

The target GS polynucleotide molecule naturally occurs in the genome ofAbutilon theophrasti, Amaranthus albus, Amaranthus chlorostachys,Amaranthus graecizans, Amaranthus hybridus, Amaranthus lividus,Amaranthus palmeri, Amaranthus rudis, Amaranthus spinosus, Amaranthusthunbergii, Ambrosia trifida, Ambrosia artemisiifolia, Chenopodiumalbum, Commelina diffusa, Convolvulus arvensis, Conyza canadensis,Lolium multiflorum, Euphorbia heterophylla, Kochia scoparia, Sorghumhalepense, and Digitaria sanguinalis and include molecules related tothe expression of a polypeptide identified as a GS, that includeregulatory molecules, cDNAs comprising coding and noncoding regions of aGS gene and fragments thereof as shown in Table 1.

Polynucleotide molecules were extracted from these plant species bymethods standard in the field, for example, total RNA is extracted usingTRIZOL® reagent (Invitrogen Corp, Carlsbad, Calif., Cat. No. 15596-018;a monophasic solution of phenol and guanidine isothiocyanate), followingthe manufacturer's protocol or modifications thereof by those skilled inthe art of polynucleotide extraction that may enhance recover or purityof the extracted RNA. Briefly, start with 1 gram of ground plant tissuefor extraction. Prealiquot 10 milliliters (mL) TRIZOL® reagent to 15 mLconical tubes. Add ground powder to tubes and shake to homogenize.Incubate the homogenized samples for 5 minutes (min) at room temperature(RT) and then add 3 mL of chloroform. Shakes tubes vigorously by handfor 15-30 seconds (sec) and incubate at RT for 3 min. Centrifuge thetubes at 7,000 revolutions per minute (rpm) for 10 min at 4 degrees C.Transfer the aqueous phase to a new 1.5 mL tube and add 1 volume of coldisopropanol. Incubate the samples for 20-30 min at RT and centrifuge at10,000 rpm for 10 min at 4 degrees C. Wash pellet with Sigma-grade 80percent ethanol. Remove the supernatant and briefly air-dry the pellet.Dissolve the RNA pellet in approximately 200 microliters of DEPC treatedwater. Heat briefly at 65 degrees C. to dissolve pellet and vortex orpipet to resuspend RNA pellet. Adjust RNA concentration to 1-2microgram/microliter.

DNA was extracted using EZNA SP Plant DNA Mini kit (Omega Biotek,Norcross Ga., Cat#D5511) and Lysing Matrix E tubes (Q-Biogen, Cat#6914),following the manufacturer's protocol or modifications thereof by thoseskilled in the art of polynucleotide extraction that may enhance recoveror purity of the extracted DNA. Briefly, aliquot ground tissue to aLysing Matrix E tube on dry ice, add 800 μl Buffer SP1 to each sample,homogenize in a bead beater for 35-45 sec, incubate on ice for 45-60sec, centrifuge at >14000 rpm for 1 min at RT, add 10 microliter RNase Ato the lysate, incubate at 65° C. for 10 min, centrifuge for 1 min atRT, add 280 μl Buffer SP2 and vortex to mix, incubate the samples on icefor 5 min, centrifuge at >10,000 g for 10 min at RT, transfer thesupernatant to a homogenizer column in a 2 ml collection tube,centrifuge at 10,000 g for 2 min at RT, transfer the cleared lysate intoa 1.5 ml microfuge tube, add 1.5 volumes Buffer SP3 to the clearedlysate, vortex immediately to obtain a homogeneous mixture, transfer upto 650 μl supernatant to the Hi-Bind column, centrifuge at 10,000 g for1 min, repeat, apply 100 μl 65° C. Elution Buffer to the column,centrifuge at 10,000 g for 5 min at RT.

Next-generation DNA sequencers, such as the 454-FLX (Roche, Branford,Conn.), the SOLiD (Applied Biosystems,), and the Genome Analyzer(HiSeq2000, Illumina, San Diego, Calif.) were used to providepolynucleotide sequence from the DNA and RNA extracted from the planttissues. Raw sequence data is assembled into contigs. The contigsequence is used to identify trigger molecules that can be applied tothe plant to enable regulation of the gene expression.

The target DNA sequence isolated from genomic (gDNA) and coding DNA(cDNA) from the various weedy plant species for the GS gene and theassembled contigs were set forth in SEQ ID NOs: 1-59 and Table 1.

Example 2 Polynucleotides of the Invention Related to the TriggerMolecules

The gene sequences and fragments of Table 1 were divided into 200polynucleotide (200-mer) lengths with 25 polynucleotide overlappingregions as in SEQ ID NOs: 37-1056. These polynucleotides are tested toselect the most efficacious trigger regions across the length of anytarget sequence. The trigger polynucleotides are constructed as sense oranti-sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids andcombined with an organosilicone based transfer agent to provide apolynucleotide preparation. The polynucleotides are combined into setsof two to three polynucleotides per set, using 4-8 nmol of eachpolynucleotide. Each polynucleotide set is prepared with the transferagent and applied to a plant or a field of plants in combination with aGS inhibitor containing herbicide, or followed by a GS inhibitortreatment one to three days after the polynucleotide application, todetermine the effect on the plant's susceptibility to a GS inhibitor.The effect is measured as stunting the growth and/or killing of theplant and is measured 8-14 days after treatment with the polynucleotideset and GS inhibitor. The most efficacious sets are identified and theindividual polynucleotides are tested in the same methods as the setsare and the most efficacious single 200-mer identified. The 200-mersequence is divided into smaller sequences of 50-70-mer regions with10-15 polynucleotide overlapping regions and the polynucleotides testedindividually. The most efficacious 50-70-mer is further divided intosmaller sequences of 25-mer regions with a 12 to 13 polynucleotideoverlapping region and tested for efficacy in combination with GSinhibitor treatment. By this method it is possible to identify anoligonucleotide or several oligonucleotides that are the mostefficacious trigger molecule to effect plant sensitivity to a GSinhibitor or modulation of a GS gene expression. The modulation of GSgene expression is determined by the detection of GS siRNA moleculesspecific to a GS gene or by an observation of a reduction in the amountof GS RNA transcript produced relative to an untreated plant or bymerely observing the anticipated phenotype of the application of thetrigger with the GS inhibiting herbicide. Detection of siRNA can beaccomplished, for example, using kits such as mirVana (Ambion, AustinTex.) and mirPremier (Sigma-Aldrich, St Louis, Mo.).

The target DNA sequence isolated from genomic (gDNA) and coding DNA(cDNA) from the various weedy plant species for the GS gene and theassembled contigs w forth in SEQ ID NOs: 1-59 were divided intopolynucleotide fragments as set forth in SEQ ID NOs: 60-1444.

The gene sequences and fragments of Table 1 were compared and 21-mers ofcontiguous polynucleotides were identified that had homology across thevarious GS gene sequences. The purpose is to identify trigger moleculesthat are useful as herbicidal molecules or in combination with a GSinhibitor herbicide across a broad range of weed species. The SEQ IDNOs: 1444-2045 sequences represent the 21-mers that are present in theGS gene of at least two of the weed species of Table 1. It iscontemplated that additional 21-mers can be selected from the sequencesof Table 1 that are specific for a single weed species or a few weedsspecies within a genus or trigger molecules that are at least 18contiguous nucleotides, at least 19 contiguous nucleotides, at least 20contiguous nucleotides or at least 21 contiguous nucleotides in lengthand at least 85 percent identical to a GS gene sequence selected fromthe group consisting of SEQ ID NOs: 1-59 or fragment thereof.

By this method it is possible to identify an oligonucleotide or severaloligonucleotides that are the most efficacious trigger molecule toeffect plant sensitivity to GS inhibitor or modulation of GS geneexpression. The modulation of GS gene expression is determined by thedetection of GS siRNA molecules specific to GS gene or by an observationof a reduction in the amount of GS RNA transcript produced relative toan untreated plant or by merely observing the anticipated phenotype ofthe application of the trigger with the GS inhibitor containingherbicide. Detection of siRNA can be accomplished, for example, usingkits such as mirVana (Ambion, Austin Tex.) and mirPremier(Sigma-Aldrich, St Louis, Mo.).

The target DNA sequence isolated from genomic (gDNA) and coding DNA(cDNA) from the various weedy plant species for the GS gene and theassembled contigs as set forth in SEQ ID NOs: 1-59 were divided intofragments as set forth in SEQ ID NOs: 1444-2045.

Example 3 Methods Used in the Invention Related to Treating Plants orPlant Parts with a Topical Mixture of the Trigger Molecules

Glyphosate-sensitive Palmer amaranth (A. palmeri R-22) plants were grownin the greenhouse (30/20 C day/night T; 14 hour photoperiod) in 4 inchsquare pots containing Sun Grog Redi-Earth and 3.5 kg/cubic meterOsmocote® 14-14-14 fertilizer. Palmer amaranth plants at 5 to 10 cm inheight were treated with a mixture of 5 short (40-mer) single-strandantisense single strand DNA polynucleotides (ssDNA) targeting GS codingsequence at 4 nm (nanomole) each, formulated in 20 millimolar sodiumphosphate buffer (pH 6.8) containing 2 percent ammonium sulfate and 1percent Silwet L-77. Three pools of ssDNA polynucleotides were tested.One pool named the GS CpGS1 contained 5 polynucleotides (SEQ ID NOs:2046-2050) that were selected to target the gene encoding thechloroplastic targeted glutamine synthetase1 gene. The second pool namedGS CytGS1 contained 5 polynucleotides (SEQ ID NOs: 2051-2055) that wereselected to target the cytosolic glutamine synthetase1 gene. The thirdpool (GS Mix) was a combination of ssDNA trigger molecules, two eachfrom the GS CpGS1 and GS CytGS1 pool plus one ssDNA (SEQ ID NOs: 2056)targeting a cytosolic glutamine synthetase2 gene. Plants are treatedmanually by pipetting 10 μL of polynucleotide solution on four fullyexpanded mature leaves, for a total of 40 microliters of solution perplant. Three days after the trigger polynucleotide treatment, the plantswere treated with Ignite® rate (Bayer Cropscience) at 1/32× (23grams/hectare) field rate. There were four replications of eachtreatment. Plant growth and development was visually rated sixteen daysafter herbicide treatment to determine the efficacy of thepolynucleotide pool and herbicide treatments. The result shown in Table2 as the average percent efficacy observed of the four replicationsrelative to the untreated control.

TABLE 2 ssDNA trigger polynucleotide activity on Palmer Amaranth,percent efficacy Formulation control GS CpGS1 GS CytGS1 GS Mix 3 19 1536

Example 4 A Method to Control Weeds in a Field

A method to control weeds in a field comprises the use of triggerpolynucleotides that can modulate the expression of a GS gene in one ormore target weed plant species. In SEQ ID NOs: 1444-2045, an analysis ofGS gene sequences from twenty-two plant species provided a collection of21-mer polynucleotides that can be used in compositions to affect thegrowth or develop or sensitivity to GS inhibitor herbicide to controlmultiple weed species in a field. A composition containing 1 or 2 or 3or 4 or more of the polynucleotides of SEQ ID NOs: 1444-2045 wouldenable broad activity of the composition against the multiple weedspecies that occur in a field environment.

The method includes creating a composition that comprises componentsthat include at least one polynucleotide of SEQ ID NOs: 1444-2045 or anyother effective gene expression modulating polynucleotide essentiallyidentical or essentially complementary to SEQ ID NOs:1-59 or fragmentthereof, a transfer agent that mobilizes the polynucleotide into a plantcell and a GS inhibiting herbicide and optionally a polynucleotide thatmodulates the expression of an essential gene and optionally a herbicidethat has a different mode of action relative to a GS inhibitor. Thepolynucleotide of the composition includes a dsRNA, ssDNA or dsDNA or acombination thereof. A composition containing a polynucleotide can havea use rate of about 1 to 30 grams or more per acre depending on the sizeof the polynucleotide and the number of polynucleotides in thecomposition. The composition may include one or more additionalherbicides as needed to provide effective multi-species weed control. Afield of crop plants in need of weed plant control is treated by sprayapplication of the composition. The composition can be provided as atank mix, a sequential treatment of components (generally thepolynucleotide followed by the herbicide), a simultaneous treatment ormixing of one or more of the components of the composition from separatecontainers. Treatment of the field can occur as often as needed toprovide weed control and the components of the composition can beadjusted to target specific weed species or weed families.

TABLE 1 Glutamine synthetase gene sequences isolated from various weedspecies SEQ ID NO SPECIES TYPE LENGTH 1 Amaranthus palmeri cDNAContig1759 2 Amaranthus palmeri gDNAContig 8486 3 Amaranthus palmerigDNAContig 6862 4 Amaranthus rudis cDNAContig 1618 5 Amaranthus rudiscDNAContig 1550 6 Amaranthus rudis gDNAContig 2000 7 Amaranthus rudisgDNAContig 208 8 Ambrosia trifida cDNAContig 1723 9 Ambrosia trifidagDNAContig 1000 10 Ambrosia trifida gDNAContig 841 11 Conyza canadensiscDNAContig 1955 12 Conyza canadensis gDNAContig 8676 13 Conyzacanadensis gDNAContig 8635 14 Euphorbia heterophylla cDNAContig 1550 15Euphorbia heterophylla gDNAContig 3777 16 Euphorbia heterophyllagDNAContig 1755 17 Commelina diffusa cDNAContig 1587 18 Commelinadiffusa gDNAContig 6900 19 Digitaria sanguinalis cDNAContig 1535 20Digitaria sanguinalis gDNAContig 7358 21 Kochia scoparia cDNAContig 91822 Kochia scoparia cDNAContig 867 23 Kochia scoparia cDNAContig 360 24Kochia scoparia gDNAContig 5248 25 Kochia scoparia gDNAContig 3994 26Kochia scoparia gDNAContig 2204 27 Kochia scoparia gDNAContig 135 28Lolium multiflorum cDNAContig 1673 29 Lolium multiflorum cDNAContig 82030 Lolium multiflorum gDNAContig 1888 31 Lolium multiflorum gDNAContig1737 32 Lolium multiflorum gDNAContig 975 33 Lolium multiflorumgDNAContig 781 34 Lolium multiflorum gDNAContig 766 35 Loliummultiflorum gDNAContig 575 36 Lolium multiflorum gDNAContig 455 37Abutilon theophrasti cDNAContig 1270 38 Abutilon theophrasti gDNAContig1252 39 Abutilon theophrasti gDNAContig 885 40 Abutilon theophrastigDNAContig 1075 41 Amaranthus albus cDNAContig 1603 42 Amaranthuschlorostachys cDNAContig 514 43 Amaranthus chlorostachys cDNAContig 114044 Amaranthus graecizans cDNAContig 1691 45 Amaranthus hybriduscDNAContig 1883 46 Amaranthus lividus cDNAContig 1683 47 Amaranthusspinosus cDNAContig 1743 48 Amaranthus thunbergii cDNAContig 1702 49Amaranthus viridis cDNAContig 1744 50 Euphorbia heterophylla gDNAContig4893 51 Sorghum halepense cDNAContig 1581 52 Convolvulus arvensiscDNAContig 710 53 Chenopodium album cDNAContig 1276 54 Ambrosiaartemisiifolia gDNAContig 671 55 Euphorbia heterophylla gDNAContig 304756 Euphorbia heterophylla gDNAContig 2153 57 Euphorbia heterophyllagDNAContig 946 58 Euphorbia heterophylla gDNAContig 375 59 Euphorbiaheterophylla gDNAContig 459

1.-31. (canceled)
 32. A method for potentiating activity of a glutaminesynthetase (GS) inhibitor herbicide in a plant comprising (a) applyingto a surface of the plant a composition comprising at least onenon-transcribable polynucleotide and a transfer agent, wherein thenon-transcribable polynucleotide is from 18 to about 700 nucleotides inlength and is identical or complementary to at least 18 contiguousnucleotides of a glutamine synthetase (GS) gene sequence or an RNAtranscript of the GS gene sequence, wherein the GS gene sequence isselected from the group consisting of SEQ ID NOs: 1-4, 6, 7, 9, 10, 14,16, 17, 20, 21, 23, 27-49, 51-59, and a polynucleotide fragment thereof,and wherein the transfer agent conditions the surface of the plant forpermeation by the non-transcribable polynucleotide, and (b) applying theGS inhibitor herbicide to the plant; whereby the non-transcribablepolynucleotide permeates the interior of the plant and inducessuppression of the GS gene, thereby potentiating activity of the GSinhibitor herbicide in the plant.
 33. The method of claim 32, whereinthe at least one non-transcribable polynucleotide is (a) at least onepolynucleotide selected from the group consisting of sense ssDNA,anti-sense ssDNA, sense ssRNA, anti-sense ssRNA, dsRNA, dsDNA, anddsDNA/RNA hybrids; (b) at least one polynucleotide selected from thegroup consisting of SEQ ID NOs: 60-389, 392-529, 532-593, 596-689,692-785, 790-859, 862-877, 880-991, 996-1069, 1072-1215, 1218-1241,1244-1263, 1266-1297, 1300-1443, and a fragment thereof; (c) at leastone polynucleotide selected from the group consisting of SEQ ID NOs:1444-2045 and a fragment thereof; (d) at least one polynucleotideselected from the group consisting of SEQ ID NOs: 2046-2056 and afragment thereof; or (e) any combination of two or morenon-transcribable polynucleotides, each being from 18 to about 700nucleotides in length and identical or complementary to at least 18contiguous nucleotides of a GS gene sequence or an RNA transcript of theGS gene sequence, wherein the GS gene sequence is selected from thegroup consisting of SEQ ID NOs: 1-4, 6, 7, 9, 10, 14, 16, 17, 20, 21,23, 27-49, 51-59, and a polynucleotide fragment thereof.
 34. The methodof claim 32, wherein the transfer agent is an organosilicone surfactantcomposition or compound contained therein.
 35. The method of claim 32,wherein the composition further comprises the GS inhibitor herbicide.36. The method of claim 35, wherein the GS inhibitor herbicide isselected from the group consisting of glufosinate-ammonium andbialaphos.
 37. The method of claim 35, wherein the composition furthercomprises one or more herbicides different from the GS inhibitorherbicide.
 38. The method of claim 35, wherein the composition furthercomprises a co-herbicide.
 39. The method of claim 38, wherein theco-herbicide is selected from the group consisting of amide herbicides,arsenical herbicides, benzothiazole herbicides, benzoylcyclohexanedioneherbicides, benzofuranyl alkylsulfonate herbicides, cyclohexene oximeherbicides, cyclopropylisoxazole herbicides, dicarboximide herbicides,dinitroaniline herbicides, dinitrophenol herbicides, diphenyl etherherbicides, dithiocarbamate herbicides, glycine herbicides, halogenatedaliphatic herbicides, imidazolinone herbicides, inorganic herbicides,nitrile herbicides, organophosphorus herbicides, oxadiazoloneherbicides, oxazole herbicides, phenoxy herbicides, phenylenediamineherbicides, pyrazole herbicides, pyridazine herbicides, pyridazinoneherbicides, pyridine herbicides, pyrimidinediamine herbicides,pyrimidinyloxybenzylamine herbicides, quaternary ammonium herbicides,thiocarbamate herbicides, thiocarbonate herbicides, thiourea herbicides,triazine herbicides, triazinone herbicides, triazole herbicides,triazolone herbicides, triazolopyrimidine herbicides, uracil herbicides,and urea herbicides.
 40. The method of claim 35, wherein the compositionfurther comprises a pesticide.
 41. The method of claim 40, wherein thepesticide is selected from the group consisting of insecticides,fungicides, nematicides, bactericides, acaricides, growth regulators,chemosterilants, semiochemicals, repellents, attractants, pheromones,feeding stimulants, and biopesticides.
 42. The method of claim 32,wherein the plant is selected from the group consisting of Abutilontheophrasti, Amaranthus albus, Amaranthus chlorostachys, Amaranthusgraecizans, Amaranthus hybridus, Amaranthus lividus, Amaranthus palmeri,Amaranthus rudis, Amaranthus spinosus, Amaranthus thunbergii, Ambrosiatrifida, Ambrosia artemisiifolia, Chenopodium album, Commelina diffusa,Convolvulus arvensis, Conyza canadensis, Lolium multiflorum, Euphorbiaheterophylla, Kochia scoparia, Sorghum halepense, and Digitariasanguinalis.
 43. A method of reducing growth, development, orreproductive ability in a plant, comprising treating a plant with a GSinhibitor herbicide in combination with (a) at least onenon-transcribable polynucleotide that is from 18 to about 700nucleotides in length and is identical or complementary to at least 18contiguous nucleotides of a glutamine synthetase (GS) gene sequence oran RNA transcript of the GS gene sequence, wherein the GS gene sequenceis selected from the group consisting of SEQ ID NOs: 1-4, 6, 7, 9, 10,14, 16, 17, 20, 21, 23, 27-49, 51-59, and a polynucleotide fragmentthereof, and (b) an organosilicone surfactant, whereby growth,development, or reproductive ability is reduced in the treated plantcompared to a control plant treated with the GS inhibitor herbicidealone.
 44. The method of claim 43, wherein the at least onenon-transcribable polynucleotide is (a) at least one polynucleotideselected from the group consisting of sense ssDNA, anti-sense ssDNA,sense ssRNA, anti-sense ssRNA, dsRNA, dsDNA, and dsDNA/RNA hybrids; (b)at least one polynucleotide selected from the group consisting of SEQ IDNOs: 60-389, 392-529, 532-593, 596-689, 692-785, 790-859, 862-877,880-991, 996-1069, 1072-1215, 1218-1241, 1244-1263, 1266-1297,1300-1443, and a fragment thereof; (c) at least one polynucleotideselected from the group consisting of SEQ ID NOs: 1444-2045 and afragment thereof; (d) at least one polynucleotide selected from thegroup consisting of SEQ ID NOs: 2046-2056 and a fragment thereof; or (e)any combination of two or more non-transcribable polynucleotides, eachbeing from 18 to about 700 nucleotides in length and identical orcomplementary to at least 18 contiguous nucleotides of an GS genesequence or an RNA transcript of the GS gene sequence, wherein the GSgene sequence is selected from the group consisting of SEQ ID NOs: 1-4,6, 7, 9, 10, 14, 16, 17, 20, 21, 23, 27-49, 51-59, and a polynucleotidefragment thereof.
 45. The method of claim 43, wherein the GS inhibitorherbicide is selected from the group consisting of glufosinate-ammoniumand bialaphos.
 46. The method of claim 43, wherein the compositionfurther comprises a co-herbicide or a pesticide.
 47. The method of claim43, wherein the plant is selected from the group consisting of Abutilontheophrasti, Amaranthus albus, Amaranthus chlorostachys, Amaranthusgraecizans, Amaranthus hybridus, Amaranthus lividus, Amaranthus palmeri,Amaranthus rudis, Amaranthus spinosus, Amaranthus thunbergii, Ambrosiatrifida, Ambrosia artemisiifolia, Chenopodium album, Commelina diffusa,Convolvulus arvensis, Conyza canadensis, Lolium multiflorum, Euphorbiaheterophylla, Kochia scoparia, Sorghum halepense, and Digitariasanguinalis.
 48. A recombinant expression cassette comprising a promoterfunctional in a microbe and operably linked to a polynucleotide that is18 to about 700 nucleotides in length and is identical or complementaryto at least 18 contiguous nucleotides of a glutamine synthetase (GS)gene sequence or an RNA transcript of the GS gene sequence, wherein theGS gene sequence is selected from the group consisting of SEQ ID NOs:1-4, 6, 7, 9, 10, 14, 16, 17, 20, 21, 23, 27-49, 51-59, and apolynucleotide fragment thereof.
 49. A method of making a recombinantpolynucleotide comprising harvesting polynucleotides from a microbe thatexpresses the recombinant expression cassette of claim
 48. 50. Anherbicidal composition comprising (a) the recombinant polynucleotidesprovided by the method of claim 49, (b) an organosilicone surfactant,and (c) a GS inhibitor herbicide.